Biology subjects

Webber, B. R.

Publications and source records attributed to Webber, B. R..

4 recordsLinked to original sources

bioRxiv2018

GeneWeld: a method for efficient targeted integration directed by short homology

Choices for genome engineering and integration involve high efficiency with little or no target specificity or high specificity with low activity. Here, we describe a targeted integration strategy, called GeneWeld, and a vector series for gene tagging, pGTag (plasmids for Gene Tagging), which promote highly efficient and precise targeted integration in zebrafish embryos, pig fibroblasts, and human cells utilizing the CRISPR/Cas9 system. Our work demonstrates that in vivo targeting of a genomic locus of interest with CRISPR/Cas9 and a donor vector containing as little as 24 to 48 base pairs of homology directs precise and efficient knock-in when the homology arms are exposed with a double strand break in vivo. Our results suggest that the length of homology is not important in the design of knock-in vectors but rather how the homology is presented to a double strand break in the genome. Given our results targeting multiple loci in different species, we expect the accompanying protocols, vectors, and web interface for homology arm design to help streamline gene targeting and applications in CRISPR and TALEN compatible systems.

genetics↗

bioRxiv2018

A Genetically Engineered Primary Human Natural Killer Cell Platform for Cancer Immunotherapy

Tumors can evade natural killer (NK) cells by activating inhibitory pathways. We therefore have developed a highly efficient CRISPR/Cas9-based method for editing the genome of peripheral blood human NK cells (PB-NKs) to knock out ADAM17 and PD1 or knock-in genes using recombinant AAV6. Our method allows editing of PB-NKs at efficiencies reaching 90%, equivalent to methods reported for primary human T cells. Moreover, we demonstrate that ADAM17 and PD1 KO PB-NKs have significantly improved activity, cytokine production, and cancer cell cytotoxicity. Our platform represents a feasible method for generating engineered NK cells as a universal therapeutic for cancer immunotherapy.

immunology↗

bioRxiv2018

Highly Multiplexed Genome Engineering Using CRISPR/Cas9 gRNA Arrays

The CRISPR/Cas9 system is an RNA guided nuclease system that evolved as a mechanism of adaptive immunity in bacteria. This system has been adopted for numerous genome engineering applications in research and recently, therapeutics. The CRISPR/Cas9 system has been largely implemented by delivery of Cas9 as protein, RNA, or plasmid along with a chimeric crRNA-tracrRNA guide RNA (gRNA) under the expression of a pol III promoter, such as U6. Using this approach, multiplex genome engineering has been achieved by delivering several U6-gRNA plasmids targeting multiple loci. However, this approach is limiting due to the efficiently of delivering multiple plasmids to a single cell at one time. To augment the capability and accessibility of multiplexed genome engineering, we developed an efficient golden gate based method to assemble gRNAs linked by optimal Csy4 ribonuclease sequences to deliver up to 10 gRNAs as a single gRNA array transcript. Here we report the optimal expression of our guide RNA array under a strong pol II promoter. This system can be implemented alongside the myriad of CRISPR applications, allowing users to model complex biological processes requiring numerous gRNAs.

bioengineering↗

bioRxiv2017

Rapid DNA Replication Origin Licensing Protects Stem Cell Pluripotency

Complete and robust human genome duplication requires loading MCM helicase complexes at many DNA replication origins, an essential process termed origin licensing. Licensing is restricted to G1 phase of the cell cycle, but G1 length varies widely among cell types. Using quantitative single cell analyses we found that pluripotent stem cells with naturally short G1 phases load MCM much faster than their isogenic differentiated counterparts with long G1 phases. During the earliest stages of differentiation towards all lineages, MCM loading slows concurrently with G1 lengthening, revealing developmental control of MCM loading. In contrast, ectopic Cyclin E overproduction uncouples short G1 from fast MCM loading. Rapid licensing in stem cells is caused by accumulation of the MCM loading protein, Cdt1. Prematurely slowing MCM loading in pluripotent cells not only lengthens G1 but also accelerates differentiation. Thus, rapid origin licensing is an intrinsic characteristic of stem cells that contributes to pluripotency maintenance.

cell biology↗